Presymptomatic screening to detect early-stage cancer reduces cancer-related mortality and treatment-related morbidity. Although many cancers can be treated and cured if they are diagnosed while tumors are still localized, most cancers are not detected until after they have invaded the surrounding tissue or metastasized to distant sites. For example, only 50% of breast cancers, 56% of prostate cancers and 35% of colorectal cancers are localized at the time of diagnosis, see Watkins, B., Szaro, R., Ball, S., Knubovets, T., Briggman, J., Hlavaty, J. J., Kusinitz, F., Stieg, A., and Wu, Y. (2001) Detection of early-stage cancer by serum protein analysis. American Laboratory. June, 32-36. incorporated herein by reference in its entirety. The situation is much worse for other, less treatable types of cancer. For example, about 80% of pancreatic cancers are already metastatic at the time of diagnosis which results in 1-year survival rate after diagnosis of about 19% and 5-year survival rate of about 4%. Similar 5-year survival rates (<5%) were reported for hepatocellular carcinoma. As therapeutic options for cancer treatment increase, early detection of cancer becomes important for improving prognosis.
In recent years, several serum protein markers have been developed for certain types of cancer. For example, prostate specific antigen (PSA), a glycoprotein secreted by prostate cells that is found in serum in prostate pathologies, is currently used as a tumor marker for prostate cancer. Other protein markers for cancer diagnostics and monitoring are alpha-fetoprotein for hepatocellular carcinoma and testicular cancer, NMP22 for bladder cancer, catecholamines for neuroblastoma, immunoglobulins for multiple myeloma, carcinoembryonic antigen (CEA) for colorectal cancer, HER-2, CA 15-3 and CA 27-29 for breast cancer, CA 125 for ovarian cancer, CA19-9 for pancreatic cancer, see Keesee et. al. Crit. Rev. Eukaryotic Gene Expr, 1996, 6(2&3): 189-214; Diamandis, Clin. Lab. News 1996, 22: 235-239, Stein et. al. J. Urol 1998, 160(3, pt 1):645-659. Although the development of the serum markers facilitated the clinical management of certain types of cancer, the assays of these biomarkers are neither sensitive nor specific enough for use as the sole screening method for cancer diagnostics. Thus, it is highly desirable to develop new cancer-related biomarkers that will be more sensitive and more specific to detect recurrence and metastases at the earliest stages for both diagnosing and monitoring cancer progression.
Methods of developing new cancer-related biomarkers were suggested based on the difference in glycosylation in glycoproteins from cancer patients and healthy controls. For example, Block et. al. was comparing glycosylation profiles in immunoglobulin G (IgG) depleted sera from hepatitis B virus infected subjects (humans and woodchucks) with hepatocellular carcinoma and from respective healthy controls to identify particular glycoproteins with glycosylation changes as cancer-related biomarkers, see Block, T. M., Comunale, M. A., Lowman, M., Steel, L. F., Romano, P. R., Fimmel, C., Tennant, B. C., London, W. T., Evans, A. A., Blumberg, B. S., Dwek, R. A., Mattu, T. S. and Mehta, A. S. (2005). “Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans.” Proc Natl Acad Sci USA 102: 779-84, incorporated herein by reference. Particular differences in glycosylation profiles of purified glycoproteins between diseased patients and healthy controls can serve themselves as markers of the disease. For example, a clear correlation between rheumatoid arthritis and the percentage of the galactosylation on N-glycans released from purified immunoglobulin G (IgG) has been established in Parekh et al., see “Association of Rheumatoid Arthritis and Primary Osteoarthritis with Changes in the Glycosylation Pattern of Total Serum IgG,” Nature, 316, pp. 452-457, 1985, incorporated herein by reference in its entirety. Alterations in glycosylation profiles of purified glycoproteins were also reported for certain types of cancer. For example, glycosylation was found to be different for glycans released from purified PSA from seminal plasma and from purified PSA secreted by the tumor prostate cell line LNCaP, see Peracaula R, Tabarés G, Royle L, Harvey D J, Dwek R A, Rudd, P M, de Llorens R. (2003). Altered glycosylation pattern allows the distinction between Prostate Specific Antigen (PSA) from normal and tumor origins, Glycobiology, 13, 457-470, incorporated herein by reference in its entirety. Completely different glycosylation profiles were found for pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3), see Peracaula R, Royle L, Tabarés G, Mallorquí-Fernández G, Barrabés S, Harvey D, Dwek R A, Rudd, P M, de Llorens R. (2003) “Glycosylation of human pancreatic ribonuclease: differences between normal and tumour states”, Glycobiology, 13, 227-244, incorporated herein by reference in its entirety. Thus, glycosylation analysis can be a powerful tool for identifying cancer-related biomarkers, however, currently used methods involve purifying glycoproteins, a step which can be time consuming and which can require a large amount of sample material from patients. Accordingly, it is highly desirable to develop methods for identifying cancer-related glycosylation markers and related methods for diagnosing and monitoring cancer that would not comprise purifying glycoproteins. Performing glycosylation analysis on whole, i.e. not depleted and not purified, samples can be particularly beneficial for cancer diagnostics and monitoring. Although differences in the glycosylation profile can be associated with the presence in samples of cancer patients of glycoproteins specifically associated with cancer, such as alpha-fetoprotein (see e.g. Johnson, P. J., T. C. Poon, et al. (2000). “Structures of disease-specific serum alpha-fetoprotein isoforms.” Br J Cancer 83(10): 1330-7; and Chan, M. H., M. M. Shing, et al. (2000). “Alpha-fetoprotein variants in a case of pancreatoblastoma.” Ann Clin Biochem 37 (Pt 5): 681-5), many other tumor glycoproteins, i.e. glycoproteins that are not specific inflammatory markers of cancer, can be expected to carry altered glycosylation because glycosylation pathways are usually disturbed in tumor cells, see e.g. “Effects of N-Glycosylation on in vitro Activity of Bowes Melanoma and Human Colon Fibroblast Derived Tissue Plasminogen Activator” Art Wittwer, Susan Howard, Linda S. Carr, Nikos K. Harakas, Joseph Feder Raj B. Parekh, Pauline M. Rudd, Raymond A. Dwek and Thomas W. Rademacher Biochemistry, 1989, 28, 7662-7669; “N-Glycosylation and in vitro Enzymatic Activity of Human Recombinant Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells and a Murine Cell line” Raj B. Parekh, Raymond A. Dwek, Pauline M. Rudd, Jerry R. Thomas, T. W. Rademacher, T. Warren, T. C. Wun, B. Herbert, B. Reitz, M. Palmier, T. Ramabhadran and D. C. Teimeir Biochemistry 1989, 28, 7670-7679, both incorporated herein in their entirety. Based on the above, performing detailed glycosylation analysis on samples of whole body fluid or body tissue, without isolating or purifying specific glycoproteins, can be expected to identify glycosylation markers of cancer amplified compared with glycosylation analysis of purified glycoproteins.